This is U.S. National phase under 35 U.S.C. xc2xa7 371 of International Application PCT/EP98/02465, filed Apr. 23, 1998, which claims priority of the Netherlands application 1005884, filed April 1997.
The present invention relates to a new regulatory system for inducible expression of genes based on lambdoid promoters. The invention further relates to a regulatory replicon and a method for producing heterologous proteins.
In order to enable production of human or animal proteins in sufficient quantities, the gene which codes for the protein is usually cloned in the bacteria Escherichia coli. This bacteria has a high synthesis capacity and is well characterized at molecular level. Bacterial regulation signals are also required for expression of the cloned gene in the bacterial host.
It has been found that the strongest regulation signals for E. coli do not originate from the bacteria itself but from the bacteria-challenging bacteriophages. There exist so-called non-temperate and temperate phages.
The first type are the phages with unregulated promoters. Genes under the control of such promoters are continuously expressed. This results in a high protein production, which can be detrimental or even lethal to the host bacteria.
The other type, the so-called temperate phages, can insert their DNA in a non-active form in the host genome and therefore co-replicate passively with this host genome. By induction of particular promoters the host is stimulated to produce phage protein or, in the case of expression vectors based on phage promoters, to produce the heterologous protein. As long as there is no induction, expression from the promoter is shut off by means of repressor molecules binding cooperatively to the promoter. The promoters of temperate phages are among the strongest, but also the best expressed and controllable promoters from E. coli known (Lanzer and Bujard (1988); Knaus and Bujard (1988)).
The combination of intrinsic strength and superior regulation make these promoters preferable to other regulated or non-regulated E. coli promoters for obtaining heterologous expression on industrial scale.
The best known and prototype phage from the group of temperate phages is the E. coli phage xcex. There are many xcex-related or lambdoid phages such as 21, xcfx8680, xcfx8681, 82, 424, 434, P22, etc. These phages usually have a different immunity, inter alia through the use of different promoter sequences, repressor molecules and operator sequences.
Many expression plasmids for heterologous protein production which are used in E. coli based on the xcexPL promoter. The xcexPL promoter is very strong and can be well regulated. The best known and most controllable regulation mechanism makes use of a thermosensitive mutant of the original repressor molecule. Induction of protein synthesis from the promoter can in this case be started by increasing the temperature from 28xc2x0 C. to 42xc2x0 C. The repressor molecule is deactivated by this temperature increase. However, this higher temperature can also be unfavorable for production of many proteins because the protein, instead of remaining soluble, then precipitates for the greater part in the form of so-called inclusion bodies, wherein it loses its activity.
Inclusion bodies are in fact an aggregate of incorrectly folded polypeptide chains. It is a phenomenon which is observed on both laboratory scale and industrial scale when an attempt is made to produce large quantities of a specific protein in E. coli. Inclusion bodies can per se be separated quite easily from the other cellular proteins in one step. However, after isolation of the inclusion bodies the protein must first be denatured by means of for instance urea or guanidine hydrochloride and then slowly refolded into the natural spatial structure. This refolding of the protein from inclusion bodies is not always successful and generally results in a considerable loss of material and entails extra costs in the scale-up process due to an increase in the number of steps in the final processing. The frequent occurrence of inclusion bodies has resulted in it not always being possible to fully utilize the potential of an economically advantageous expression host for heterologous protein production.
It has been found that the formation of inclusion bodies can sometimes be prevented by reducing the fermentation temperature. The reason therefor may be either that the lower temperature has a different effect on the folding of the overproduced protein or that there are fewer newly synthesized protein molecules per unit of time and volume.
When protein synthesis at a lower temperature is desired, it is no longer possible to use the currently existing and much used temperature induction in combination with the strong and well regulated promoters derived from phage lambda and related promoters.
It is therefore the object of the present invention to provide a simple, well controllable regulation system for strong and highly repressible promoters derived from lambdoid phages, with which induction at a lower temperature becomes possible.
This is achieved by the invention with a regulation system for expression vectors, comprising a lambdoid promoter, a gene coding for a repressor for the lambdoid promoter and a gene coding for an antirepressor of the repressor, which antirepressor gene is under the control of an inducible promoter. This promoter can originate from a. gene other than the antirepressor gene itself and is preferably inducible at lower temperatures.
The regulation of the heterologous protein expression can now be controlled by the expression of the antirepressor. The absence or presence of the antirepressor determines the suppression or activation, respectively, of the promoter of the protein to be produced. The presence or absence of the antirepressor is in turn regulated by whether or not the promoter of the antirepressor is induced.
Regulation of the antirepressor gene can occur in different ways. Use can thus be made for instance of a promoter regulatable by lactose, arabinose or the absence of amino acids or of any other regulatable promoter.
The different components of the regulation system according to the invention can be located on the chromosome of the host as well as on one or more individual replicons, such as plasmids. In a particular embodiment the antirepressor gene can lie on a regulatory plasmid together with the gene coding for the repressor of the promoter of the antirepressor gene. Such a regulatory plasmid can then be combined with any random expression vehicle containing the heterologous gene and its promoter and repression system. Optionally the repression system of the promoter of the heterologous gene can also be situated on the regulatory vehicle. All components can also lie on different replicons.
In a preferred embodiment of the invention the antirepressor is the ant of the lambdoid phage P22. Ant is coded in the immI region of phage P22 from Salmonella typhimurium and engages in a non-covalent interaction with the C-terminal part of the P22c2 repressor and thereby prevents the dimerization of the c2 repressor required for repressor activity and thereby binding to the operator.
In a preferred embodiment of the regulation system according to the invention the expression of this anti-repressor is under the control of an inducible promoter such as PN25/O2. Repression of the PN25/O2 promoter takes place for instance by means of the lacI repressor of E. coli. The induction of this promoter is based on derepression and preferably takes place by means of administering IPTG.
The regulation system according to the invention is a flexible system wherein according to a preferred embodiment the induction of the desired heterologous protein synthesis can take place in two ways. On the one hand the production of the antirepressor can already be initiated at low temperature by adding IPTG, which leads to derepression of the lambdoid promoter. It is also possible to use the same bacterial culture, wherein the conventional temperature-dependent induction can still be applied.
The regulation system can be applied with any expression vector derived from lambda. As regulatable promoter of the heterologous gene the xcexPL promoter is particularly recommended, but the invention is certainly not limited thereto. The xcexPR promoter or any lambdoid promoter which can be repressed by a repressor with sufficient homology in the C-terminal region to P22c2 to be recognized by ant can also be used.
The principle of the invention, i.e. regulating a promoter inducible by a repression system by means of an antirepressor to be expressed in regulated manner, can of course also be applied within the scope of the present invention in configurations other than those specifically described herein.
The invention further relates to a regulatory replicon, comprising a gene coding for an antirepressor, which antirepressor gene is under the control of an inducible promoter. The replicon can further comprise a gene coding for a repressor of the inducible promoter of the antirepressor gene. A gene which codes for a repressor for a lambdoid promoter can moreover also be present in the replicon.
In a preferred embodiment a replicon according to the invention comprises the gene coding for the P22ant protein of S. tyohimurium, under the control of the PN25/O2 promoter, the lacIq gene under the control of the pLacIq promoter and the gene coding for the cI857 repressor.
A preferred embodiment of a regulatory replicon according to the invention is shown in FIGS. 1 and 3. Both figures show the plasmid, designated herein pICA2. FIG. 3 shows the general structure and FIG. 1 the restriction map. The construction of this plasmid is described in the examples.
In an alternative embodiment of the replicon according to the invention the replicon can further comprise the regulation signals, including the lambdoid promoter, required for expression of a heterologous gene.
In such an embodiment there are not therefore two separate vectors for expression and regulation, but only one.
The invention moreover relates to an expression system, comprising a regulatory replicon and an expression vector derived from phage lambda. Examples of expression vectors derived from phage lambda are pLT10T or pLR10T.
Finally, the invention relates to a method for producing a gene product in a heterologous host, comprising providing a culture of a host comprising a heterologous sequence which codes for the gene product, wherein the expression of the heterologous sequence is under the control of a regulation system which at least consists of a lambdoid promoter operably linked to the heterologous sequence, a gene coding for a repressor for the lambdoid promoter and a gene coding for an antirepressor, which antirepressor gene is operably linked to an inducible promoter, and of inducing the promoter of the antirepressor gene. If the inducible promoter of the antirepressor gene is the PN25/O2 promoter, it can be induced by adding IPTG to the culture.